CD-Laboratory for Biotechnology of Glycerol, Muthgasse 18, 1190 Vienna, Austria.
Department of Biotechnology, BOKU-VIBT, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
FEMS Microbiol Lett. 2019 Feb 1;366(4). doi: 10.1093/femsle/fnz022.
The yeast Yarrowia lipolytica represents a future microbial cell factory for numerous applications in a bio-based economy. Outstanding feature of this yeast is the metabolic flexibility in utilising various substrates (sugars, fatty acids, glycerol, etc.). The potential of wild-type isolates of Y. lipolytica to convert glycerol into various value-added compounds is attracting attention of academia and industry. However, the already established tools for efficient engineering of the metabolism of Y. lipolytica are often dependent on genetic features like auxotrophic markers. With the present work we want to introduce a new set of vectors for metabolic engineering strategies, including CRISPR/Cas9 technology. The system is based on GoldenMOCS, a recently established rapid Golden Gate cloning strategy applicable in multiple organisms. We could show that our new GoldenMOCS plasmids are suitable for the extrachromosomal overexpression of the gene glycerol kinase (GUT1) in wild-type isolates of Y. lipolytica resulting in enhanced conversion of glycerol to erythritol and citric acid. Moreover, a GoldenMOCS plasmid for CRISPR/Cas9 mediated genome editing has been designed, which facilitates single gene knock-outs with efficiencies between 6% and 25% in strains with genetic wild-type background.
酵母解脂耶氏酵母代表了未来微生物细胞工厂在生物经济中的众多应用。这种酵母的突出特点是在利用各种底物（糖、脂肪酸、甘油等）方面具有代谢灵活性。野生型解脂耶氏酵母分离株将甘油转化为各种高附加值化合物的潜力引起了学术界和工业界的关注。然而，已经建立的用于有效工程改造解脂耶氏酵母代谢的工具通常依赖于营养缺陷型标记等遗传特征。在本工作中，我们希望引入一组新的载体，用于代谢工程策略，包括 CRISPR/Cas9 技术。该系统基于 GoldenMOCS，这是一种新建立的适用于多种生物体的快速 Golden Gate 克隆策略。我们证明了我们的新 GoldenMOCS 质粒适合在野生型解脂耶氏酵母分离株中外源过表达甘油激酶（GUT1）基因，从而增强了甘油向赤藓糖醇和柠檬酸的转化。此外，还设计了用于 CRISPR/Cas9 介导的基因组编辑的 GoldenMOCS 质粒，在具有遗传野生型背景的菌株中，单基因敲除效率在 6%至 25%之间。
